Viral variants

ABSTRACT

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus variants exhibiting complete or partial resistance to nucleoside analogues and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity. The present invention further contemplates assays for detecting such viral variants which assays are useful in monitoring anti-viral therapeutic regimes and in developing new or modified vaccines directed against viral agents and in particular hepatitis B virus variants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of patent CooperationInternational Application PCT/AU00/00294, filed 7 Apr. 2000, whichapplication claims priority from Australian Patent Application No.PP9679 filed 9 Apr. 1999.

The present invention relates generally to viral variants exhibitingreduced sensitivity to particular agents and/or reduced interactivitywith immunological reagents. More particularly, the present invention isdirected to hepatitis B virus variants exhibiting complete or partialresistance to nucleoside analogues and/or reduced interactivity withantibodies to viral surface components including reduced sensitivity.The present invention further contemplates assays for detecting suchviral variants which assays are useful in monitoring anti-viraltherapeutic regimes and in developing new or modified vaccines directedagainst viral agents and in particular hepatitis B virus variants.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications numerically referred to inthis specification are collected at the end of the description.

Specific mutations in an amino acid sequence are represented herein asAXaa₁nXaa₂″ where Xaa₁ is the original amino acid residue beforemutation, n is the residue number and Xaa₂ is the mutant amino acid. Theabbreviation “Xaa” may be the three letter or single letter (i.e. “X”)code. The amino acid residues for Hepatitis B virus DNA polymerase arenumbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD(SEQ ID NO:10)) being residue number 550.

Hepatitis B virus (HBV) can cause debilitating disease conditions andcan lead to acute liver failure. HBV is a DNA virus which replicates viaan RNA intermediate and utilizes reverse transcription in itsreplication strategy (1). The HBV genome is of a complex nature having apartially double stranded DNA structure with overlapping open readingframes encoding surface, core, polymerase and X genes. The complexnature of the HBV genome is represented in FIG. 1.

The presence of an HBV DNA polymerase has led to the proposition thatnucleoside analogues could act as effective anti-viral agents. Examplesof nucleoside analogues currently being tested are penciclovir and itsoral form famciclovir (2, 3, 4, 5), lamivudine (6,7). Adefovir has beenshown to have effective anti-HBV activity in vitro. Generally, suchnucleotide analogues are used in conjunction with Hepatitis Bimmunoglobulin (HBIG) therapy. There is potential for such agents to beused in the treatment of chronic HBV infection.

Penciclovir has been shown to have potent inhibitory activity againstduck HBV DNA synthesis in vitro and has been shown to inhibit HBV DNApolymerase-reverse transcriptase activity in vitro (8,9). Similarly,oral famiciclovir has been demonstrated to inhibit intra-hepaticreplication of duck HBV virus in vivo (10). In man, famciclovir has beenshown to reduce HBV DNA replication in a patient with severe hepatitis Bfollowing orthotopic liver transplantation (OLT) (11).

In work leading up to the present invention, nucleoside analogueantiviral therapy was used to control severe post-OLT recurrence of HBVinfection (12). Long term therapy is mandatory where patients areimmunosuppressed and the rate of HBV replication is very high. However,under such conditions, as with any long term chemotherapy of infectiousagents, there is a potential for development of resistance or reducedsensitivity to the therapeutic agents employed. In addition, somepatients do not respond to famciclovir pre-OLT. This may be due topatients not metabolising famciclovir or patients infected with afamciclovir-resistant HBV variant.

In accordance with the present invention, the inventors have identifiedvariants of HBV with mutations in the HBV DNA polymerase gene which tovarying extents reduce the sensitivity of HBV to nucleoside analogues.The identification of these HBV variants is important for thedevelopment of assays to monitor nucleoside analogue therapeutic regimesand to screen for agents which can mask the effects of the mutation,i.e. in the development of new vaccines. In addition, since the HBVgenome comprises a series of overlapping open reading frames, anucleotide mutation in one open reading frame can affect translationproducts in another open reading frame. In further accordance with thepresent invention, the inventors have observed mutations which reducethe interactivity of immunological reagents, such as antibodies andimmune cells, to viral surface components. Such viral variants arereferred to herein as “escape mutants” since they potentially escapeexisting immunological memory.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word Acomprise≅, or variations such as Acomprises≅ or Acomprising≅,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier, i.e. SEQ ID NO:1, SEQ ID NO:2, etc. A sequence listing isprovided after the claims.

One aspect of the present invention is directed to a variant of anisolated DNA virus which replicates via an RNA intermediate wherein saidvariant comprises a nucleotide mutation in a gene encoding a DNApolymerase resulting in at least one amino acid addition, substitutionand/or deletion to said DNA polymerase.

Another aspect of the present invention provides a variant of anisolated DNA virus which replicates via an RNA intermediate wherein saidvariant comprises a nucleotide mutation in a gene encoding a viralsurface component resulting in at least one amino acid addition,substitution and/or deletion in said viral surface component.

Still a further aspect of the present invention is directed to a variantof an isolated DNA virus which replicates via an RNA intermediate atleast wherein said variant comprises a nucleotide mutation in anoverlapping portion of at least two open reading frames resulting in anamino acid addition, substitution and/or deletion to translationproducts of said open reading frames.

Still yet a further aspect of the present invention provides an HBVvariant comprising a mutation in the nucleotide sequence encoding a DNApolymerase resulting in an amino acid addition, substitution and/ordeletion in said DNA polymerase in one or more amino acids as set forthin Formula I (SEQ ID NO:7):S Z₁ L S W L S L D V S A A F Y H Z₂ P L H P A A M P H L L Z₃ G S S G LZ₄ R Y V A R LS S Z₅ S Z₆ Z₇ X N Z₈ Q Z₉ Z₁₀ X X X Z₁₁ L H Z₁₂ Z₁₃ C S R Z₁₄ L Y V S LZ₁₅ L L Y Z₁₆ T Z₁₇G Z₁₈ K L H L Z₁₉ Z₂₀ H P I Z₂₁ L G F R K Z₂₂ P M G Z₂₃ G L S P F L L AQ F T S A I Z₂₄ Z₂₅Z₂₆ Z₂₇ Z₂₈ R A F Z₂₉ H C Z₃₀ Z₃₁ F Z₃₂ Y M* D D Z₃₃ V L G A Z₃₄ Z₃₅ Z₃₆Z₃₇ H Z₃₈ E Z₃₉ L Z₄₀Z₄₁ Z₄₂ Z₄₃ Z₄₄ Z₄₅ Z₄₆ L L Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R W G Y S L NF M G Y Z₅₀I G  FORMULA Iwherein:X is any amino acid;Z₁ is N or D;Z₂ is I or P;Z₃ is I or V;Z₄ is S or D;Z₅ is T or N;Z₆ is R or N;Z₇ is N or I;Z₈ is N or Y or H;Z₉ is H or Y;Z₁₀ is G or R;Z₁₁ is D or N;Z₁₂ is D or N;Z₁₃ is S or Y;Z₁₄ is N or Q;Z₁₅ is L or M;Z₁₆ is K or Q;Z₁₇ is Y or F;Z₁₈ is R or W;Z₁₉ is Y or L;Z₂₀ is S or A;Z₂₁ is I or V;Z₂₂ is I or L;Z₂₃ is V or G;Z₂₄ is C or L;Z₂₅ is A or S;Z₂₆ is V or M;Z₂₇ is V or T;Z₂₈ is R or C;Z₂₉ is F or P;Z₃₀ is L or V;Z₃₁ is A or V;Z₃₂ is S or A;Z₃₃ is V or L or M;Z₃₄ is K or R;Z₃₅ is S or T;Z₃₆ is V or G;Z₃₇ is Q or E;Z₃₈ is L or S or R;Z₃₉ is S or F;Z₄₀ is F or Y;Z₄₁ is T or A;Z₄₂ is A or S;Z₄₃ is V or I;Z₄₄ is T or C;Z₄₅ is N or S;Z₄₆ is F or V;Z₄₇ is S or D;Z₄₈ is L or V;Z₄₉ is N or Q;Z₅₀ is V or I; andM* is amino acid 550provided said mutation is not in the YMDD (SEQ ID NO: 10) motif of the Cdomain alone, and wherein said variant exhibits decreased sensitivity toa nucleoside analogue.

Another aspect of the present invention contemplates an HBV variantcomprising a mutation in the nucleotide sequence encoding a viralsurface component resulting in an amino acid addition, substitutionand/or deletion in said viral surface component in a regioncorresponding to the amino acid sequence set forth in Formula I whereinsaid variant exhibits decreased interactivity of immunological reagentsto said viral surface component.

Yet another aspect of the present invention provides an HBV variantcomprising a mutation in the nucleotide sequence encoding a viralsurface component resulting in an amino acid addition, substitutionand/or addition in said viral surface component in a region defined byamino acids 67–226 of the HBV surface antigen or functionally equivalentregion wherein said variant exhibits decreased interactivity ofimmunological reagents to said viral surface component.

Still another aspect of the present invention provides an HBV variantcomprising a mutation in an overlapping open reading frame in its genomewherein said mutation is in a region defined by one or more of domains Fand A through E of HBV DNA polymerase provided that it is not in theYMDD (SEQ ID NO:10) motif of the C domain alone; and in the overlappingregion corresponding to amino acids 67–226 of HBV surface antigen; andwherein said variant exhibits decreased sensitivity to a nucleotideanalogue and exhibits decreased interactivity to immunological reagentsspecific to HBV surface antigens.

Still yet another aspect of the present invention contemplates a methodfor determining the potential for an HBV to exhibit reduced sensitivityto a nucleoside analogue, said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV DNA polymerase resulting in at leastone amino acid substitution, deletion and/or addition in any one or moreof domains F and A through E or a region proximal thereto of said DNApolymerase wherein the presence of such a mutation is an indication ofthe likelihood of resistance to said nucleoside analogue. Even stillanother aspect of the present invention provides a method fordetermining the potential for an HBV to exhibit reduced interactivity toantibody to HBV surface antigen, said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV surface antigen resulting in at leastone amino acid substitution, deletion and/or addition in amino acids 67to 226 of said surface antigen or a region proximal thereto of saidsurface antigen wherein the presence of such a mutation is an indicationof the likelihood of reducing interactivity of said antibodies to saidmutated surface antigen.

Another aspect of the present invention contemplates method fordetermining whether an HBV isolate encodes a variant DNA polymerase,said method comprising determining the amino acid sequence of its DNApolymerase directly or via a nucleotide sequence and comparing same tothe amino acid sequence below (SEQ ID NO:7):S Z₁ L S W L S L D V S A A F Y H Z₂ P L H P A A M P H L L Z₃ G S S G LZ₄ R Y V A R LS S Z₅ S Z₆ Z₇ X N Z₈ Q Z₉ Z₁₀ X X X Z₁₁ L H Z₁₂ Z₁₃ C S R Z₁₄ L Y V S LZ₁₅ L L Y Z₁₆ T Z₁₇G Z₁₈ K L H L Z₁₉ Z₂₀ H P I Z₁₁ L G F R K Z₂₂ P M G Z₂₃ G L S P F L L AQ F T S A I Z₂₄ Z₂₅Z₂₆ Z₂₇ Z₂₈ R A F Z₂₉ H C Z₃₀ Z₃₁ F Z₃₂ Y M D D Z₃₃ V L G A Z₃₄ Z₃₅ Z₃₆Z₃₇ H Z₃₈ E Z₃₉ L Z₄₀Z₄₁ Z₄₂ Z₄₃ Z₄₄ Z₄₅ Z₄₆ L L Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R W G Y S L NF M G Y Z₅₀I G  FORMULA Iwherein:X is any amino acid;Z₁ is N or D;Z₂ is I or P;Z₃ is I or V;Z₄ is S or D;Z₅ is T or N;Z₆ is R or N;Z₇ is N or I;Z₈ is N or Y or H;Z₉ is H or Y;Z₁₀ is G or R;Z₁₁ is D or N;Z₁₂ is D or N;Z₁₃ is S or Y;Z₁₄ is N or Q;Z₁₅ is L or M;Z₁₆ is K or Q;Z₁₇ is Y or F;Z₁₈ is R or W;Z₁₉ is Y or L;Z₂₀ is S or A;Z₂₁ is I or V;Z₂₂ is I or L;Z₂₃ is V or G;Z₂₄ is C or L;Z₂₅ is A or S;Z₂₆ is V or M;Z₂₇ is V or T;Z₂₈ is R or C;Z₂₉ is F or P;Z₃₀ is L or V;Z₃₁ is A or V;Z₃₂ is S or A;Z₃₃ is V or L or M;Z₃₄ is K or R;Z₃₅ is S or T;Z₃₆ is V or G;Z₃₇ is Q or E;Z₃₈ is L or S or R;Z₃₉ is S or F;Z₄₀ is F or Y;Z₄₁ is T or A;Z₄₂ is A or S;Z₄₃ is V or I;Z₄₄ is T or C;Z₄₅ is N or S;Z₄₆ is F or V;Z₄₇ is S or D;Z₄₈ is L or V;Z₄₉ is N or Q;Z₅₀ is V or I; andM* is amino acid 550.

Yet another aspect of the present invention is directed to an isolatedvariant HBV surface antigen or a recombinant or derivative form thereofor a chemical equivalent thereof wherein said surface antigen or itsrecombinant or derivative form or its chemical equivalent exhibits analtered immunological profile compared to a surface antigen from areference HBV.

Still another aspect of the present invention provides an HBV vaccinecontaining one or more HBV variants carrying mutations which alter thesurface antigen (not including G145R).

Yet another aspect of the present invention provides a compositioncomprising a variant HBV or an HBV surface antigen from said variant HBVor a recombinant or derivative form thereof or its chemical equivalentand one or more pharmaceutically acceptable carriers or diluents.

Still yet another aspect of the present invention provides a use of avariant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in agene encoding a DNA polymerase resulting in at least one amino acidaddition, substitution and/or deletion to said DNA polymerase in themanufacture of a medicament for the treatment and/or prophylaxis ofhepatitis.

Even still yet another aspect of the present invention provides a use ofa variant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in agene encoding a viral surface component resulting in at least one aminoacid addition, substitution and/or deletion in said viral surfacecomponent in the manufacture of a medicament for the treatment and/orprophylaxis of hepatitis.

Another aspect of the present invention provides a use of a variant ofan isolated DNA virus which replicates via an RNA intermediate whereinsaid variant comprises a nucleotide mutation in an overlapping portionof at least two open reading frames resulting in an amino acid addition,substitution and/or deletion to translation products of said openreading frames in the manufacture of a medicament for the treatmentand/or prophylaxis of hepatitis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing the partially doublestranded DNA HBV genome showing the overlapping open reading framesencoding surface (S), core (C), polymerase (P) and X gene.

FIG. 2 is a representation showing conserved regions of domain A to E(underlined) of HBV (SEQ ID NO:7). M in YMDD (SEQ ID NO:10) isdesignated amino acid number 550. * indicates greater than three aminoacid possibilities at this position of the consensus sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Accordingly, one aspect of the present invention is directed to avariant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in agene encoding a DNA polymerase resulting in at least one amino acidaddition, substitution and/or deletion to said DNA polymerase.

Another aspect of the present invention provides a variant of anisolated DNA virus which replicates via an RNA intermediate wherein saidvariant comprises a nucleotide mutation in a gene encoding a viralsurface component resulting in at least one amino acid addition,substitution and/or deletion in said viral surface component.

Still a further aspect of the present invention is directed to a variantof an isolated DNA virus which replicates via an RNA intermediate atleast wherein said variant comprises a nucleotide mutation in anoverlapping portion of at least two open reading frames resulting in anamino acid addition, substitution and/or deletion to translationproducts of said open reading frames.

Preferably, the DNA virus is a hepatitis virus or a related virus and ismost preferably HBV.

A Arelated virus≅ in accordance with the present invention is onerelated at the genetic, immunological, epidemiological and/orbiochemical levels.

Preferably, the mutation in the DNA polymerase results in decreasedsensitivity of the HBV to a nucleoside analogue.

Preferably, the mutation in the viral surface component reduces theinteractivity of immunological reagents such as antibodies and immunecells to the viral surface component. Most preferably, the viral surfacecomponent is a viral surface antigen. The reduction in the interactivityof immunological reagents to a viral surface component generallyincludes the absence of immunological memory to recognize orsubstantially recognize the viral surface component.

A viral variant may, in accordance with a preferred aspect of thepresent invention, carry mutation only in the DNA polymerase or thesurface antigen or may carry a mutation in both molecules. The termAmutation≅ is to be read in its broadest context and includes a silentmutation not substantially affecting the normal function of the DNApolymerase or surface antigen or may be an active mutation having theeffect of selection of nucleoside analogue resistance or an escapemutant phenotype. Where multiple mutations occur in accordance with thepresent invention or where multiple phenotypes result from a singlemutation, at least one mutation must be active or the virus must exhibitat least one altered phenotype such as nucleoside analogue resistance orreduced immunological interactivity to the surface antigen.

Regions of the HBV polymerase show amino acid similarity with otherRNA-dependent DNA polymerases and RNA-dependent polymerases (13). Thepresent invention extends to all domains of the HBV DNA polymerase andin particular regions F and A through E. In this specification,reference is particularly made to the conserved regions defined by Pochet al. (13) as domains A to E (see also reference 18). Regions A to Eare defined by the amino acid sequence set forth in Formula I below:S Z₁ L S W L S L D V S A A F Y H Z₂ P L H P A A M P H L L Z₃ G S S G LZ₄ R Y V A R LS S Z₅ S Z₆ Z₇ X N Z₈ Q Z₉ Z₁₀ X X X Z₁₁ L H Z₁₂ Z₁₃ C S R Z₁₄ L Y V S LZ₁₅ L L Y Z₁₆ T Z₁₇G Z₁₈ K L H L Z₁₉ Z₂₀ H P I Z₂₁ L G F R K Z₂₂ P M G Z₂₃ G L S P F L L AQ F T S A I Z₂₄ Z₂₅Z₂₆ Z₂₇ Z₂₈ R A F Z₂₉ H C Z₃₀ Z₃₁ F Z₃₂ Y M* D D Z₃₃ V L G A Z₃₄ Z₃₅ Z₃₆Z₃₇ H Z₃₈ E Z₃₉ L Z₄₀Z₄₁ Z₄₂ Z₄₃ Z₄₄ Z₄₅ Z₄₆ L L Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R W G Y S L NF M G Y Z₅₀I G  FORMULA Iwherein:X is any amino acid;Z₁ is N or D;Z₂ is I or P;Z₃ is I or V;Z₄ is S or D;Z₅ is T or N;Z₆ is R or N;Z₇ is N or I;Z₈ is N or Y or H;Z₉ is H or Y;Z₁₀ is G or R;Z₁₁ is D or N;Z₁₂ is D or N;Z₁₃ is S or Y;Z₁₄ is N or Q;Z₁₅ is L or M;Z₁₆ is K or Q;Z₁₇ is Y or F;Z₁₈ is R or W;Z₁₉ is Y or L;Z₂₀ is S or A;Z₂₁ is I or V;Z₂₂ is I or L;Z₂₃ is V or G;Z₂₄ is C or L;Z₂₅ is A or S;Z₂₆ is V or M;Z₂₇ is V or T;Z₂₈ is R or C;Z₂₉ is F or P;Z₃₀ is L or V;Z₃₁ is A or V;Z₃₂ is S or A;Z₃₃ is V or L or M;Z₃₄ is K or R;Z₃₅ is S or T;Z₃₆ is V or G;Z₃₇ is Q or E;Z₃₈ is L or S or R;Z₃₉ is S or F;Z₄₀ is F or Y;Z₄₁ is T or A;Z₄₂ is A or S;Z₄₃ is V or I;Z₄₄ is T or C;Z₄₅ is N or S;Z₄₆ is F or V;Z₄₇ is S or D;Z₄₈ is L or V;Z₄₉ is N or Q;Z₅₀ is V or I; andM* is amino acid 550.

Preferably, the mutation results in an altered amino acid sequence inany one or more of domains F and A through E or regions proximal theretoof the HBV DNA polymerase. The present invention does not extend to amutation alone in the YMDD (SEQ ID NO:10) motif of the C domain of theHBV DNA polymerase although such a mutation is contemplated by thepresent invention if it occurs in combination with one or more mutationsin another location.

The mutation in the viral surface component is preferably in one or moreamino acid residues within the major hydrophilic regions of the protein,and in particular within the amino acid sequence 67–226 of the HBV viralsurface antigen.

According to a preferred aspect of the present invention, there isprovided an HBV variant comprising a mutation in the nucleotide sequenceencoding a DNA polymerase resulting in an amino acid addition,substitution and/or deletion in said DNA polymerase in one or more aminoacids as set forth in Formula I:S Z₁ L S W L S L D V S A A F Y H Z₂ P L H P A A M P H L L Z₃ G S S G LZ₄ R Y V A R LS S Z₅ S Z₆ Z₇ X N Z₈ Q Z₉ Z₁₀ X X X Z₁₁ L H Z₁₂ Z₁₃ C S R Z₁₄ L Y V S LZ₁₅ L L Y Z₁₆ T Z₁₇G Z₁₈ K L H L Z₁₉ Z₂₀ H P I Z₂₁ L G F R K Z₂₂ P M G Z₂₃ G L S P F L L AQ F T S A I Z₂₄ Z₂₅Z₂₆ Z₂₇ Z₂₈ R A F Z₂₉ H C Z₃₀ Z₃₁ F Z₃₂ Y M* D D Z₃₃ V L G A Z₃₄ Z₃₅ Z₃₆Z₃₇ H Z₃₈ E Z₃₉ L Z₄₀Z₄₁ Z₄₂ Z₄₃ Z₄₄ Z₄₅ Z₄₆ L L Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R W G Y S L NF M G Y Z₅₀I G  FORMULA Iwherein:X is any amino acid;Z₁ is N or D;Z₂ is I or P;Z₃ is I or V;Z₄ is S or D;Z₅ is T or N;Z₆ is R or N;Z₇ is N or I;Z₈ is N or Y or H;Z₉ is H or Y;Z₁₀ is G or R;Z₁₁ is D or N;Z₁₂ is D or N;Z₁₃ i S or Y;Z₁₄ is N or Q;Z₁₅ is L or M;Z₁₆ is K or Q;Z₁₇ is Y or F;Z₁₈ is R or W;Z₁₉ is Y or L;Z₂₀ is S or A;Z₂₁ is I or V;Z₂₂ is I or L;Z₂₃ is V or G;Z₂₄ is C or L;Z₂₅ is A or S;Z₂₆ is V or M;Z₂₇ is V or T;Z₂₈ is R or C;Z₂₉ is F or P;Z₃₀ is L or V;Z₃₁ is A or V;Z₃₂ is S or A;Z₃₃ is V or L or M;Z₃₄ is K or R;Z₃₅ is S or T;Z₃₆ is V or G;Z₃₇ is Q or E;Z₃₈ is L or S or R;Z₃₉ is S or F;Z₄₀ is F or Y;Z₄₁ is T or A;Z₄₂ is A or S;Z₄₃ is V or I;Z₄₄ is T or C;Z₄₅ is N or S;Z₄₆ is F or V;Z₄₇ is S or D;Z₄₈ is L or V;Z₄₉ is N or Q;Z₅₀ is V or I; andM* is amino acid 550provided said mutation is not in the YMDD (SEQ ID NO:10) motif of the Cdomain alone, and wherein said variant exhibits decreased sensitivity toa nucleoside analogue.

Another preferred aspect of the present invention contemplates an HBVvariant comprising a mutation in the nucleotide sequence encoding aviral surface component resulting in an amino acid addition,substitution and/or deletion in said viral surface component in a regioncorresponding to the amino acid sequence set forth in Formula I whereinsaid variant exhibits decreased interactivity of immunological reagentsto said viral surface component.

Yet another preferred aspect of the present invention relates to an HBVvariant comprising a mutation in the nucleotide sequence encoding aviral surface component resulting in an amino acid addition,substitution and/or addition in said viral surface component in a regiondefined by amino acids 67–226 of the HBV surface antigen or functionallyequivalent region wherein said variant exhibits decreased interactivityof immunological reagents to said viral surface component.

Still yet another aspect of the present invention is directed to an HBVvariant comprising a mutation in an overlapping open reading frame inits genome wherein said mutation is in a region defined by one or moreof domains F and A through E of HBV DNA polymerase provided that it isnot in the YMDD (SEQ ID NO:10) motif of the C domain alone; and in theoverlapping region corresponding to amino acids 67 to 226 of HBV surfaceantigen and wherein said variant exhibits decreased sensitivity to anucleotide analogue and exhibits decreased interactivity toimmunological reagents specific to HBV surface antigens.

One particular mutant is M550I/V which has been previously describedfollowing lamivudine treatment. The present invention does not extend tothis mutant in so far as it arises following treatment with lamuvidinealone. An M550V mutant is selected in conjunction with the mutationL526M and this is also not within the scope of the present invention.

The present invention does not extend to the following lamuvidineresistance mutations when selected by lamuvidine treatment alone,however, it does extend to these mutations when selected duringfamciclovir (FCV) treatment:

L428M, T481C, T496A, L497F, V509I, V519L, L526M, T530S, A546V, F548V,M550I, V553I, S559T, Q561H, S565A, A568T, 1570S, L575M, L581I and N584S(16, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 42, 45).

Furthermore, the present invention does not extend to the followingpublished famciclovir selected mutations:

S424T, Del 462–468, I509V, V519L, P523L, L526M, L526V, A528T, T530S,V5531, S565A, I570V and N594H (38, 39, 41, 43, 44, 46).

The viral variant exhibiting reduced interactivity to immunologicalreagents is an escape mutant since antibodies or other immunologicalresponse to HBV from a prior exposure to the virus or followingvaccination are no longer effective in targeting a viral surfacecomponent since the mutation has altered a B- and/or T-cell epitope onthe surface antigen.

Reduced or decreased sensitivity to nucleotide, analogue orimmunological agents is also encompassed by the term Aresistance≅. Theterm Aresistance≅ is used in its most general sense and includes totalresistance or partial resistance or decreased sensitivity to anucleoside analogue.

Preferably, the variants are in isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV.

Preferred mutations in the HBV DNA polymerase and/or surface antigeninclude variants selected from patients following HBV recurrencefollowing famciclovir and HBIG treatment, and patients who did notrespond to famciclovir treatment as indicated by a decreased in HBV DNAand/or viral protein.

Preferred mutations in the HBV DNA polymerase together withcorresponding mutation in the surface antigen (shown in parentheses)include one or more of the L423L/M/V (I68I/M), L423L/F (C69F/L),H436H/Y, H436Y, DEL 471–474 (DEL 117–120), S438T, W499E(D144E, G145R),1508V, V519L (E164D), L526M, S565A(S210R), N584S, N/S/H584N/K, R588R/Kand N594H such as selected in patients with HBV recurrence followingfamciclovir and HBIG treatment; and H436N/H, S463S/Y (L1091/L), V537V/I(C/W182Y/STOP), V/G560E (Y206N), S/F 565A/S(S210R/S), S/F 565A (S210R),N/Q 584H, K587R and N594H, such as selected in patients who did notrespond to famciclovir. Preferred mutations in the surface antigeninclude one or more of the following V96A, C138R, P142T/P, K160K/N andA1194G/A after only hepatitis B virus immunoglobulin (HBIG) treatment.The term ADEL≅ means Adeletion≅ and ASTOP≅ means a stop codon.

The present invention does not extend to a mutation in the Hepatitis Bsurface antigen at G145R alone or in combination with D144E (23) whenthese mutations are selected without famciclovir treatment.

Particularly preferred mutations in the HBV DNA polymerase together withcorresponding mutations in the surface antigen (shown in parentheses)include one or more of L423L/MV [I68I/M], H436H/Y, H436Y, DEL471–474[DEL117–120], W499E [D144E and G145R], V519L [E164D], N/S/H 584 N/K andR588 R/K such as selected in patients with HBV recurrence followingfamciclovir and HBIG treatment; and H436H/N, S463 S/Y [L1091/L], V537V/I [C/W 182 Y/STOP], and K587R, such as selected in non-respondingpatients following famciclovir treatment.

The identification of the variants of the present invention permits thegeneration of a range of assays to detect such variants. The detectionof such variants may be important in identifying resistant variants todetermine the appropriate form of chemotherapy and/or to monitorvaccination protocols, develop new or modified vaccine preparations.

Accordingly, another aspect of the present invention contemplates amethod for determining the potential for an HBV to exhibit reducedsensitivity to a nucleoside analogue, said method comprising isolatingDNA or corresponding mRNA from said HBV and screening for a mutation inthe nucleotide sequence encoding HBV DNA polymerase resulting in atleast one amino acid substitution, deletion and/or addition in any oneor more of domains A through E or a region proximal thereto of said DNApolymerase wherein the presence of such a mutation is an indication ofthe likelihood of resistance to said nucleoside analogue.

A further aspect of the present invention provides a method fordetermining the potential for an HBV to exhibit reduced interactivity toantibody to HBV surface antigen, said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV surface antigen resulting in at leastone amino acid substitution, deletion and/or addition in amino acids 67to 226 of said surface antigen or a region proximal thereto of saidsurface antigen wherein the presence of such a mutation is an indicationof the likelihood of reducing interactivity of said antibodies to saidmutated surface antigen.

Preferably, the assay detects one or more of the following mutations inthe HBV DNA polymerase (with the corresponding mutation in the surfaceantigen shown in parentheses): L423L/M/V (I68I/M), L423L/F (C69F/L),H436H/Y, H436Y, DEL 471–474 (DEL 117–120), S438T, W499E (D144E, G145R),1508V, V519L (E164D), L526M, S565A(S210R), N584S, N/S/H584N/K, R588R/Kand N594H such as selected in patients with HBV recurrence followingfamciclovir and HBIG treatment; and H436N/H, S463S/Y (L109I/L), V537V/I(C/W182Y/STOP), V/G560E (Y206N), S/F 565A/S(S210R/S), S/F 565A (S210R),N/Q 584H, K587R and N594H, such as selected in patients who did notrespond to famciclovir. Mutations may also be detected in the surfaceantigen including one or more of: V96A, C138R, P142T/P, K160K/N andA194G/A such as after Hepatitis B virus immunoglobulin (HBIG) treatment.

More particularly, the assay detects one or more of the followingmutations in the HBV DNA polymerase (with corresponding mutations in thesurface antigen shown in parentheses): L423L/MV [I68I/M], H436H/Y,H436Y, DEL471–474 [DEL117-120], W499E [D144E and G145R], V519L [E164D],N/S/H 584 N/K and R588 R/K such as selected in patients with HBVrecurrence following famciclovir and HBIG treatment; and H436H/N, S463S/Y [L109I/L], V537 V/I [C/W 182 Y/STOP], and K587R, such as selected innon-responding patients following famciclovir treatment.

The DNA or corresponding RNA may be assayed or alternatively the DNApolymerase or surface antigen may be screened for the mutation.

The detection according to this aspect of the invention may be anynucleic acid-based detection means, for example nucleic acidhybridisation techniques or polymerase chain reaction (PCR). Theinvention further encompasses the use of different assay formats of saidnucleic acid-based detection means, including restriction fragmentlength polymorphism (RFLP), amplified fragment length polymorphism(AFLP), single-strand chain polymorphism (SSCP), amplification andmismatch detection (AMD), interspersed repetitive sequence polymerasechain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) andreverse transcription polymerase chain reaction (RT-PCR), amongstothers.

The present invention extends to a range of immunologically based assaysto detect variant HBV DNA polymerase or surface antigen. These assaysare based on antibodies directed to naturally occurring HBV DNApolymerase or surface antigen which do not, or substantially do not,interact with the variant HBV DNA polymerase or surface antigen.Alternatively, antibodies to a variant HBV DNA polymerase or surfaceantigen are used which do not or substantially do not, interact withnaturally occurring HBV DNA polymerase or surface antigen.

Monoclonal or polyclonal antibodies may be used although monoclonalantibodies are preferred as they can be produced in large quantity andin a homogenous form. A wide range of immunoassay techniques areavailable such as described in U.S. Pat. Nos. 4,016,043, 4,424,279 and4,018,653.

The detection of amino acid variants of DNA polymerase is convenientlyaccomplished by reference to the consensus amino acid sequence shown inFIG. 2. The polymorphisms shown represent the variations shown invarious data bases for active pathogenic HBV strains. Where an HBVvariant comprises an amino acid different to what is represented, thensuch an isolate is considered a putative HBV variant having an alteredDNA polymerase activity.

Accordingly, another aspect of the present invention contemplates amethod for determining whether an HBV isolate encodes a variant DNApolymerase, said method comprising determining the amino acid sequenceof its DNA polymerase directly or via a nucleotide sequence andcomparing same to the amino acid sequence below:S Z₁ L S W L S L D V S A A F Y H Z₂ P L H P A A M P H L L Z₃ G S S G LZ₄ R Y V A R LS S Z₅ S Z₆ Z₇ X N Z₈ Q Z₉ Z₁₀ X X X Z₁₁ L H Z₁₂ Z₁₃ C S R Z₁₄ L Y V S LZ₁₅ L L Y Z₁₆ T Z₁₇G Z₁₈ K L H L Z₁₉ Z₂₀ H P I Z₂₁ L G F R K Z₂₂ P M G Z₂₃ G L S P F L L AQ F T S A I Z₂₄ Z₂₅Z₂₆ Z₂₇ Z₂₈ R A F Z₂₉ H C Z₃₀ Z₃₁ F Z₃₂ Y M* D D Z₃₃ V L G A Z₃₄ Z₃₅ Z₃₆Z₃₇ H Z₃₈ E Z₃₉ L Z₄₀Z₄₁ Z₄₂ Z₄₃ Z₄₄ Z₄₅ L L Z₄₆ L Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R W G Y S LN F M G Y Z₅₀I Gwherein:X is any amino acid;Z₁ is N or D;Z₂ is I or P;Z₃ is I or V;Z₄ is S or D;Z₅ is T or N;Z₆ is R or N;Z₇ is N or I;Z₈ is N or Y or H;Z₉ is H or Y;Z₁₀ is G or R;Z₁₁ is D or N;Z₁₂ is D or N;Z₁₃ is S or Y;Z₁₄ is N or Q;Z₁₅ is L or M;Z₁₆ is K or Q;Z₁₇ is Y or F;Z₁₈ is R or W;Z₁₉ is Y or L;Z₂₀ is S or A;Z₂₁ is I or V;Z₂₂ is I or L;Z₂₃ is V or G;Z₂₄ is C or L;Z₂₅ is A or S;Z₂₆ is V or M;Z₂₇ is V or T;Z₂₈ is R or C;Z₂₉ is F or P;Z₃₀ is L or V;Z₃₁ is A or V;Z₃₂ is S or A;Z₃₃ is V or L or M;Z₃₄ is K or R;Z₃₅ is S or T;Z₃₆ is V or G;Z₃₇ is Q or E;Z₃₈ is L or S or R;Z₃₉ is S or F;Z₄₀ is F or Y;Z₄₁ is T or A;Z₄₂ is A or S;Z₄₃ is V or I;Z₄₄ is T or C;Z₄₅ is N or S;Z₄₆ is F or V;Z₄₇ is S or D;Z₄₈ is L or V;Z₄₉ is N or Q;Z₅₀ is V or I; andM* is amino acid 550.

The present invention further contemplates agents which mask thenucleoside analogue resistance mutation. Such agents will beparticularly useful in long term treatment by nucleoside analogues. Theagents may be DNA or RNA or proteinaceous or non-proteinaceous chemicalmolecules. Natural product screening such as from plants, coral andmicroorganisms is also contemplated as a useful potential source ofmasking agents. The agents maybe in isolated form or in the form of apharmaceutical composition and may be administered sequentially orsimultaneously with the nucleoside analogue.

The present invention further extends to an isolated surface componentfrom the HBV variants herein described. More particularly, the presentinvention provides an isolated surface antigen or a recombinant formthereof or derivative or chemical equivalent thereof. The isolatedsurface component and, more particularly, isolated surface antigen orits recombinant, derivative or chemical equivalents are useful in thedevelopment of biological compositions such as vaccine formulations.

Accordingly, another aspect of the present invention is directed to anisolated variant HBV surface antigen or a recombinant or derivative formthereof or a chemical equivalent thereof wherein said surface antigen orits recombinant or derivative form or its chemical equivalent exhibitsan altered immunological profile compared to a surface antigen from areference HBV. More particularly, the present invention provides anisolated variant HBV surface antigen or a recombinant or derivative formthereof or a chemical equivalent thereof wherein said HBV surfaceantigen or its recombinant or derivative form or its chemical equivalentcomprises an amino acid sequence with a single or multiple amino acidsubstitution, addition and/or deletion or a truncation compared to anHBV surface antigen from a reference HBV and wherein a neutralisingantibody directed to a reference HBV exhibits no or reduced neutralisingactivity to an HBV carrying said variant HBV surface antigen.

The present invention particularly contemplates an HBV vaccinecontaining one or more of the mutations which alter the surface antigen(not including G145R). Preferred mutations in the surface antigen andthe HBV vaccine include one or more of I68I/M, C69F/L, H436Y, DEL117–120, D144E, E164D, S210R, such as selected in patients with HBVrecurrence following famciclovir and HBIG treatment; and L109I/L,C/W182Y/STOP, Y206N, S210R/S and S210R; such as selected in patients whodo not respond to famciclovir. Variants carrying mutations in thesurface antigen at V96A, C138R, P142T/P, K160K/N and/or A194G/A areparticularly preferred.

The term “isolated” means the same as it does in relation to an isolatedHBV variant.

As stated above, the present invention extends to derivatives andchemical equivalents (i.e. analogues) of the HBV surface component.Derivatives include single or multiple amino acid substitutions,additions and/or deletions to the HBV surface antigen. AAdditions≅ toamino acid sequences include fusions with other peptides, polypeptidesor proteins or fusions to nucleotide sequences including fusions toother viral components.

Analogues of the variant HBV surface antigen contemplated hereininclude, but are not limited to, modification to side chains,incorporating of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other methods which impose conformational constraints on theproteinaceous molecule or their analogues. These types of modificationsare useful in stabilizing the immunointeractive molecules for use indiagnostic assays or in therapeutic protocols.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration withtetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid, contemplated herein is shown below in Table 1. The inclusionof such unnatural amino acids or other derivations described herein mayassist in stabilising the molecule in a vaccine composition.

TABLE 1 Non-conventional amino acid Code α-aminobutyric acid Abuα-amino-α-methylbutyrate Mgabu aminocyclopropane- Cpro carboxylateaminoisobutyric acid Aib aminonorbornyl- Norb carboxylatecyclohexylalanine Chexa cyclopentylalanine Cpen D-alanine Dal D-arginineDarg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamicacid Dglu D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysineDlys D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-prolineDpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine DtyrD-valine Dval D-α-methylalanine Dmala D-α-methylarginine DmargD-α-methylasparagine Dmasn D-α-methylaspartate Dmasp D-α-methylcysteineDmcys D-α-methylglutamine Dmgln D-α-methylhistidine DmhisD-α-methylisoleucine Dmile D-α-methylleucine Dmleu D-α-methyllysineDmlys D-α-methylmethionine Dmmet D-α-methylornithine DmornD-α-methylphenylalanine Dmphe D-α-methylproline Dmpro D-α-methylserineDmser D-α-methylthreonine Dmthr D-α-methyltryptophan DmtrpD-α-methyltyrosine Dmty D-α-methylvaline Dmval D-N-methylalanine DnmalaD-N-methylarginine Dnmarg D-N-methylasparagine DnmasnD-N-methylaspartate Dnmasp D-N-methylcysteine Dnmcys D-N-methylglutamineDnmgln D-N-methylglutamate Dnmglu D-N-methylhistidine DnmhisD-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysineDnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine DnmornN-methylglycine Nala N-methylaminoisobutyrate NmaibN-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine NleuD-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvalineDnmval γ-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine EtgL-homophenylalanine Hphe L-α-methylarginine Marg L-α-methylaspartateMasp L-α-methylcysteine Mcys L-α-methylglutamine MglnL-α-methylhistidine Mhis L-α-methylisoleucine Mile L-α-methylleucineMleu L-α-methylmethionine Mmet L-α-methylnorvaline MnvaL-α-methylphenylalanine Mphe L-α-methylserine Mser L-α-methyltryptophanMtrp L-α-methylvaline Mval N-(N-(2,2-diphenylethyl) Nnbhmcarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane L-N-methylalanine Nmala L-N-methylarginine NmargL-N-methylasparagine Nmasn L-N-methylaspartic acid NmaspL-N-methylcysteine Nmcys L-N-methylglutamine Nmgln L-N-methylglutamicacid Nmglu L-N-methylhistidine Nmhis L-N-methylisolleucine NmileL-N-methylleucine Nmleu L-N-methyllysine Nmlys L-N-methylmethionineNmmet L-N-methylnorleucine Nmnle L-N-methylnorvaline NmnvaL-N-methylornithine Nmorn L-N-methylphenylalanine NmpheL-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine NmthrL-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr L-N-methylvalineNmval L-N-methylethylglycine Nmetg L-N-methyl-t-butylglycine NmtbugL-norleucine Nle L-norvaline Nva α-methyl-aminoisobutyrate Maibα-methyl-γ-aminobutyrate Mgabu a-methylcyclohexylalanine Mchexaa-methylcylcopentylalanine Mcpen α-methyl-α-napthylalanine Manapα-methylpenicillamine Mpen N-(4-aminobutyl)glycine NgluN-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine NornN-amino-α-methylbutyrate Nmaabu α-napthylalanine Anap N-benzylglycineNphe N-(2-carbamylethyl)glycine Ngln N-(carbamylmethyl)glycine NasnN-(2-carboxyethyl)glycine Nglu N-(carboxymethyl)glycine NaspN-cyclobutylglycine Ncbut N-cycloheptylglycine Nchep N-cyclohexylglycineNchex N-cyclodecylglycine Ncdec N-cylcododecylglycine NcdodN-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-cycloundecylglycine Ncund N-(2,2-diphenylethyl)glycine NbhmN-(3,3-diphenylpropyl)glycine Nbhe N-(3-guanidinopropyl)glycine NargN-(1-hydroxyethyl)glycine Nthr N-(hydroxyethyl))glycine NserN-(imidazolylethyl))glycine Nhis N-(3-indolylyethyl)glycine NhtrpN-methyl-γ-aminobutyrate Nmgabu D-N-methylmethionine DnmmetN-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine DnmpheD-N-methylproline Dnmpro D-N-methylserine Dnmser D-N-methylthreonineDnmthr N-(1-methylethyl)glycine Nval N-methyla-napthylalanine NmanapN-methylpenicillamine Nmpen N-(p-hydroxyphenyl)glycine NhtyrN-(thiomethyl)glycine Ncys penicillamine Pen L-α-methylalanine MalaL-α-methylasparagine Masn L-α-methyl-t-butylglycine MtbugL-methylethylglycine Metg L-α-methylglutamate MgluL-α-methylhomophenylalanine Mhphe N-(2-methylthioethyl)glycine NmetL-α-methyllysine Mlys L-α-methylnorleucine Mnle L-α-methylornithine MornL-α-methylproline Mpro L-α-methylthreonine Mthr L-α-methyltyrosine MtyrL-N-methylhomophenylalanine Nmhphe N-(N-(3,3-diphenylpropyl) Nnbhecarbamylmethyl)glycine

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

As stated above, these types of modifications may be important tostabilize the variant HBsAg molecule if administered to an individual orfor use as a diagnostic reagent.

Other derivatives contemplated by the present invention include a rangeof glycosylation variants from a completely unglycosylated molecule to amodified glycosylated molecule. Altered glycosylation patterns mayresult from expression of recombinant molecules in different host cells.

Another aspect of the present invention extends to the variant HBVsurface antigen molecule or its recombinant, derivative or chemical formor a variant HBV comprising said HBV surface antigen in compositionform. Such compositions are particularly useful as therapeuticcompositions and may be referred to herein interchangeably asbiological, vaccine or pharmaceutical compositions. The biologicalcompositions are particularly useful in inducing immunological memoryagainst infection by an HBV variant such as an HBV escape mutantcontrolling by administering a variant HBV surface antigen or arecombinant, derivative or chemical form thereof or an HBV comprisingsame capable of inducing an immune response including immunologicalmemory agents.

Accordingly, the present invention contemplates a composition comprisinga variant HBV or an HBV surface antigen from said variant HBV or arecombinant or derivative form thereof or its chemical equivalent. Thecomposition may be considered as a biological composition.

Generally, if an HBV is used, it is first attenuated. The biologicalcomposition according to this aspect of the present invention generallyfurther comprises one or more pharmaceutically acceptable carriersand/or diluents.

The biological composition may comprise an HBV surface antigen or likemolecule from one HBV variant or the composition may be a cocktail ofHBsAgs or like molecules from a range of HBV variants including thereferenced HBV. Similar inclusions apply where the composition comprisesan HBV.

The biological composition forms suitable for injectable use includesterile aqueous solutions (where water soluble) or sterile powders forthe extemporaneous preparation of sterile injectable solutions. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or diluent containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The preventions of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the HBsAg orlike molecule or HBV variant or reference strain in the required amountin the appropriate solvent or diluent as followed by sterilization suchas by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying technique whichyield a powder of the immunointeractive molecule plus any additionaldesired ingredient from previously sterile-filtered solution thereof.Routes of administration contemplated by the present invention includingintravenous, intraperitoneal, intrathelial, subcutaneous andintracerebral.

The biological composition of the present invention may also be given inoral, bucal, nasal spray, inhalation, patch, drip or suppository form.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the immunointeractive molecule, use thereof in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

The HBV surface antigen or like molecule or HBV variant or referencestrain will be added in a concentration effective to induce an interactimmune response against the same molecule or an HBV carrying the same oran immunologically similar molecule. For example, an effective amount ofHBV surface antigen may range from about 10 mg to about 2000 ng, or 50ng to about 1000 mg or 100 ng to about 500 mg or other suitableeffective amount. It is sometimes more convenient to express dosageamounts in terms of body weight. Accordingly, the effective amounts maybe from, for example, about 0.5 ng/kg body weight to about 500 mg/kgbody weight or an amount therebetween.

The subject invention extends to kits for assays for variant HBV. Suchkits may, for example, contain the reagents from PCR or other nucleicacid hybridisation technology or reagents for immunologically baseddetection techniques.

The present invention further contemplates a variant of an isolated DNAvirus which replicates via an RNA intermediate wherein said variantcomprises a nucleotide mutation in a gene encoding a DNA polymeraseresulting in at least one amino acid addition, substitution and/ordeletion to said DNA polymerase in the manufacture of a medicament forthe treatment and/or prophylaxis of hepatitis.

In a related embodiment, there is provided a use of a variant of anisolated DNA virus which replicates via an RNA intermediate wherein saidvariant comprises a nucleotide mutation in a gene encoding a viralsurface component resulting in at least one amino acid addition,substitution and/or deletion in said viral surface component in themanufacture of a medicament for the treatment and/or prophylaxis ofhepatitis.

In a further related embodiment, there is provide a use of a variant ofan isolated DNA virus which replicates via an RNA intermediate whereinsaid variant comprises a nucleotide mutation in an overlapping portionof at least two open reading frames resulting in an amino acid addition,substitution and/or deletion to translation products of said openreading frames in the manufacture of a medicament for the treatmentand/or prophylaxis of hepatitis.

The present invention also provides for the use of the subject HBVvariants to screen for anti-viral agents. These anti-viral agentsinhibit the virus. The term Ainhibit≅ includes antagonizing or otherwisepreventing infection, replication, assembly and/or release or anyintermediate step. Preferred anti-viral agents include nucleosideanalogues, however, the present invention extends to non-nucleosidemolecules.

Accordingly, another aspect of the present invention contemplates theuse of a variant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in agene encoding a DNA polymerase resulting in at least one amino acidaddition, substitution and/or deletion to said DNA polymerase in inscreening for an anti-viral agent capable of inhibiting said virus.

Another aspect of the present invention provides for the use of avariant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in agene encoding a viral surface component resulting in at least one aminoacid addition, substitution and/or deletion in said viral surfacecomponent in in screening for an anti-viral agent capable of inhibitingsaid virus.

Yet another aspect of the present invention is directed to the use of avariant of an isolated DNA virus which replicates via an RNAintermediate wherein said variant comprises a nucleotide mutation in anoverlapping portion of at least two open reading frames resulting in anamino acid addition, substitution and/or deletion to translationproducts of said open reading frames in in screening for an anti-viralagent capable of inhibiting said virus.

The present invention is further described by the following non-limitingExamples.

EXAMPLES

In order to identify what other mutations may be selected during FCVtherapy in the OLT setting, the inventors sequenced and analysed the HBVDNA polymerase encompassing the catalytic domains from 26 patients withhigh levels of HBV (greater than 90 pg/ml HBV DNA) undergoing FCVtherapy and 10 patients with low levels of HBV (less than 90 pg/ml HBVDNA). Multiple serial samples were analysed, including prior to therapyand pre-OLT, during the FCV response phase post-OLT, and during HBVrecurrence post OLT. The methods and results are shown in Examples 1 to7.

Example 1 Patients and Methods

Treatment Protocol

The clinical details of the FCV prophylaxis liver transplantationprotocol as previously described (19). Briefly, the aim of the study wasto compare the safety and efficacy of oral FCV and IV penciclovir inreducing the risk of hepatitis B re-infection post-OLT in livertransplant patients. The study design was a multicentre, randomized,part double-blind, part placebo controlled trial in patients with endstage liver disease requiring OLT. Patients with HBV DNA levels morethan 90 pg/ml at study entry (by hybridization) [high replicators] weretreated with both FCV and HBIG post-OLT. These high replicators weretreated with FCV (500 mg tds) to reduce HBV DNA levels prior to OLT.Famciclovir treatment was continued for 12 months post-OLT. An untreatedcontrol group of patients with HBV DNA levels less than 90 pg/ml [lowreplicators] at study entry were treated with HBIG alone post-OLT. Inthe original study, thirty six patients underwent OLT, and of these theclinical and virological outcome of twenty-two of the FCV treatedpatients has recently been presented (17 Manns). Essentially, FCVtreated high replicators who became HBV DNA undetectable prior to OLThad HBV recurrences with similar frequency as low replicators treatedwith HBIG alone.

Example 2 Patients

Twenty six patients who had HBV levels more than 90 pg/ml at study entry(high HBV replicator [HR]) were treated with FCV pre-OLT and thosepatients who responded were then treated with HBIG plus FCV post-OLT. Ofthe 19 patients who responded to FCV and subsequently went to OLT, 9 didnot have HBV recurrence 0–12 months post-OLT and 10 had HBV recurrenceby 12 months post-OLT. Of the initial 26 patients, 6 did not initiallyrespond to FCV and 1 was withdrawn from the study because of treatmentwith lamivudine. Ten patients who had HBV levels of less than 90 pg/mlat study entry (low HBV replicator [LR]) were treated only with HBIGpost-OLT. Six of these patients had no HBV recurrence at 12 monthspost-OLT and 4 patients had HBV recurrence during the 12 monthspost-OLT.

Example 3 Extraction of HBV DNA from Patient Serum

HBV DNA was extracted from a total of 90 samples from 36 patients.Aliquots of 50 ml of sera were mixed with 150 ml TE (10 mmol/LTris-HCl(pH 7.5), 2 mmol/L EDTA), 1% w/v sodium dodecyl sulfate and 1 mg/mlproteinase K, and incubated at 55° C. for 30 mins. DNA was deproteinizedby phenol/chloroform extraction, precipitated with isopropanol anddissolved in 40 ml nuclease-free water.

Example 4 PCR Amplification

Two oligonucleotide primers (Bresatec, Adelaide, Australia) were used toamplify a fragment encompassing the catalytic domain of the polymeraseprotein and the Aa≅ determinant of the surface protein. The first roundsense primer (5′-GCC TCA TTT TGT GGG TCA CCA TA-3′ SEQ ID NO:1), and theantisense primer (5′-TCT CTG ACA TAC TTT CCA AT-3′ SEQ ID NO:2) wereused in the amplification. Each reaction was carried out using 5 ml ofthe extracted DNA as template, 1.5 U of Taq polymerase (Qiagen,Melbourne Australia), 1 mmol/L of sense and antisense primers, 200mmol/L each of deoxynucleoside triphosphates, 50 mmol/L KCl, 3.5 mmol/LMgCl, 10 mmol/LTris-HCl (pH 8.3) and 0.01% w/v gelatin. PCR wasperformed by 40 cycles of denaturation (94° C. for 45 sec), annealing(55° C. for 45 sec) and extension (72° C. for 1.5 min), followed by afinal extension of 7 min (Perkin-Elmer 2400, Cetus, Norwalk, Conn.). Ifrequired, a further hemi-nested round of amplification was performedusing 2 ml of first round product as template and primer 5′ TTG GGG TGGAGC CCT CAG GTC 3′ SEQ ID NO:3 as the sense primer. The amplificationconditions were the same as the initial round except with only 25 roundsof cycling.

Example 5 Sequencing of the Polymerase/Envelope Genes of HBV DNA

Amplified products were gel purified using Geneclean II (BIO 101 Inc.,La Jolla, Calif.) and were directly sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit according to themanufacturers specifications (Perkin Elmer, Cetus Norwalk, Conn.).Electrophoresis was carried out by the Australian Genome ResearchFacility (Walter and Eliza Hall Institute, Melbourne). The PCR primerswere used as sequencing primers as well as several additional primers(5′-AAA TTC GCA GTC CCC AAC-3′ SEQ ID NO:4,5′-GAA AAT TGG TAA CAG CGG-3′SEQ ID NO:5, and 5′-GTA TCC CTC CTG TTG CTG T-3′ SEQ ID NO:6) requiredto sequence the internal regions of the PCR products. MacVector andAssemblyLIGN (MacVector version 6.0 and AssemblyLIGN, Oxford Molecular,UK) was used to analyze all automatic sequence data. The deduced aminoacid sequences were compared using the subprogram C1usta1W.

Example 6 Sequence Analysis

Sequences were analyzed by comparison of the deduced amino acid sequencewithin the polymerase gene to the published polymerase consensussequence (FIG. 2; Formula I; ref. 18). A unique amino acid change wasdefined as a change to an amino acid which is not present in the HBVpolymerase consensus sequence. Unique changes in the HBV isolates werecompared to the individual pretreatment isolate and where appropriate,sequences were compared to other pretreatment samples. Any changeswithin the conserved polymerase domains A to E were noted (where domainA includes amino acids 421–436, Domain B includes 505–529, Domain Cincludes 546–555, Domain D includes 576–586 and Domain E includes592–601). Due to the overlapping open reading frames for the genesencoding the polymerase and envelope (HBsAg), unique changes in thepolymerase gene which alter the HBsAg were noted.

The deduced amino acid sequences for the surface antigen in theoverlapping reading frame were compared to the 88 published sequences,genotypes A to F from Norder et al (20). An unique amino acid change wasdefined as a change which is not present in the published sequences(20). In addition, unique changes were compared to published amino acidchanges detected after HBIG treatment and/or vaccination (21, 22, 23,24, 25, 26). Due to the overlapping open reading frames for thepolymerase and envelope genes, unique changes in the envelope gene whichalter the polymerase were noted.

Example 7 Results Outcome Analysis

In this study, famciclovir (FCV) was given prophylactically pre-OLT topatients with end-stage liver failure due to chronic HBV infection withserum HBV DNA levels greater than 90 pg/ml, [high replicators (HR)]. Thepatients were then subsequently treated with HBIG and FCV post-OLT.Samples were taken for sequencing pretreatment with FCV, pre-OLT, duringthe FCV responding phase post-OLT and then in those cases with risingviraemia during HBV recurrence. HBV DNA sequence was examined from 36patients. These patients included 26 high replicators [HBV DNA greaterthan 90 pg/ml at study entry (HR)] who were treated with both FCV andHBIG. Of these patients, 19 responded pre-OLT, 6 did not initiallyrespond and 1 patient was withdrawn because of treatment withlamivudine. The 19 patients who responded underwent OLT and 9 of thesepatients had no HBV recurrence at 12 months post-OLT and sera wereavailable post-OLT for 7 of these patients. The remaining 10 of thesepatients had HBV recurrence. Serum samples were available for study fromseven of these patients after HBV recurrence post-OLT. The 10 patientstreated with HBIG alone had pre-OLT serum HBV DNA levels of less than 90pg/ml [low replicators (LR)]. Of these, 4 patients had HBV recurrence inthe 12 months post-OLT. Sera was available from three of these patientspre-OLT and from two patients after HBV recurrence. Six patients had noHBV recurrence at 12 months post-OLT and sera was available from five ofthese patients post-OLT.

Sequence Analysis

(A) Patients with HBV DNA Greater than 90 pg/ml (HR Group)

Pre OLT Phase

(i) FCV Responders

Nineteen of the original 26 HR patients responded to FCV. (One furtherpatient responded but was withdrawn from further analysis because ofsubsequent treatment with lamivudine.) There were 28 unique amino acidchanges throughout the region encompassing the catalytic region of thepolymerase when compared to the published sequences (Table 2). Thisincludes 9 unique mutations within the functional domains in 8 patientswho responded to FCV. These were at L423F (A domain), I508V (B domain),V/L/M553I/M (C domain), N/Q584S (D domain), N/Q584H (D domain), S592H (Edomain), N594H (E domain), N594H/Y (E domain), and M596T/M (E domain).

There were also 28 unique changes in the HBsAg in these pretreatmentisolates compared to the sequences published in Norder et al., (20)[Table 2]. Of these mutations, 8 amino acids have been previously notedeither at the same position or are the identical amino acid reportedassociated with either vaccine escape or selection after HBIG treatmentpost OLT. Three of these were at amino acid position 120(P/S/A120S/A/T/P, P/S/A120T and P/S/A120T/P) and four were at amino acidposition 134 (F/Y/I134V, F/Y/I134N/Y, F/Y/I134S and F/Y/I134N). Theother change was at D/A144E/N.

(ii) FCV Non-Responders

Six patients with high levels of HBV DNA [HR] pre-transplantation didnot respond to FCV treatment. There were 10 unique amino acid changesfrom HBV isolated from all six patients when compared to the previouslypublished polymerase consensus (Table 4). Of these, there were 4 aminoacids H436N/H, S463S/Y, V537V/I and K587R which were not present in the19 FCV responders (Table 1). The changes detected in the polymerase geneat position 463, 537, 560, and 565 (in 2 isolates) all resulted in analtered HBsAg in the overlapping reading frame (Table 4).

There were 8 unique amino acid differences in the HBsAg compared to thesequences in Norder et al (20) in 5/6 patients. These include the 5changes noted above which also resulted in a unique mutation in thepolymerase gene, P/S/A120Y/S and S204R, which resulted in a change inthe polymerase gene that appears in the polymerase consensus sequence(ie. not unique) and P217L, which did not result in a change to thepolymerase gene. One of these amino acids is located at the sameposition as a known HBIG selected variant (P120Q) at P/S/A120Y/S.

Post OLT Phase

(i) HBV Recurrence

Ten of the nineteen patients with high levels of HBV DNA at study entry[HR] had recurrence post-OLT within the first 12 months. Of thesepatients, sera were available after recurrence for sequence analysisfrom 7 patients. There were 15 unique changes in 5/7 patients comparedto the previously published consensus sequence, or in the individualpretreatment sequences from these patients, including 10 within theconserved domains (Table 5). Eight of these 15 unique amino aciddifferences (detected in 4/7 patients) were also not detected in anypretreatment samples from the 19 patients who responded to FCV pre OLT,nor in the post-OLT samples from the 9 patients treated with FCV withoutrecurrence. These were L423L/M/V (A domain), H436H/Y (A domain), H436Y(A domain), a deletion of amino acids 471–474, W499E, V519L (B domain),N584N/K (D domain) and R588R/K (Table 5). The change at V519L is thesame as previously reported after long term FCV therapy (27). The L526Mmutation which has been previously reported after long term FCV therapy(27), was also detected in this study in one sample from a patient whoresponded to FCV (Table 5) but was not detected in latter samples fromthe same patient. The H436Y mutation was seen in isolates from twodifferent patients with recurrence, and in both cases was seen as atransitory change (i.e. had reverted to the original sequence in thenext sample). A change at this amino acid position to a differentresidue was also seen in a non-responder (see above).

The 15 unique changes in the HBV polymerase were then examined todetermine if there was any alteration in the HBsAg in the overlappingreading frame (Table 5). The deletion at residues 471–474 was found toresult in a corresponding in-frame deletion in the envelope gene (aa117–120). The H436Y, H436H/Y, S483T, 1508V and L526M mutations did notresult in any change to the envelope gene sequence. The L423L/MN/Valtered the HBsAg sequence to I68I/M, the mutation at L423F/L alteredthe HBsAg to C69F/L, the V519L resulted in E164D in the HBsAg sequenceand S565A altered the HBsAg at S210R. The mutation at W499E (due to 2nucleotide changes) resulted in a change at both D144E and G145R in theHBsAg sequence (a known vaccine escape mutant) and the N584N/K, N584S,R588R/K and the N594H changes were located after the end of the HBsAggene. The HBsAg termination codon at position 226 overlaps with thecodon encoding amino acid 582 in the polymerase gene.

There were a total of 11 unique amino acid changes in HBsAg whencompared to the published sequences of Norder et al., (20) and theindividual pretreatment sequences. This includes the seven mutationslisted above which changed the HBV polymerase and P67P/Q, P67Q, R73P andM133T which did not alter the HBV polymerase. Of these 11 changes, twoin one patient have been previously reported as vaccine or HBIG escape(D144E and G145R). The 5 unique changes in the HBsAg post-OLT inpatients with HBV recurrence not detected in any pretreatment sample arelisted in Table 6.

(ii) HBV Non-Recurrence

Nine of the HR patients treated with FCV and HBIG did not have HBVrecurrence in the 12 months following OLT and sera was available from 7patients for sequence analysis. There were 12 unique changes in threepatients compared to the previously published HBV polymerase consensussequence that were not present in the individual's pretreatment sample.These included 5 changes in the functional domains and were at L423L/F(A domain), A432V (A domain), R466K, N477T, N485N/K, G498E, L526M (Bdomain), T530S, N572K, F573Y, L577L/M/V (D domain) and L593G/V/L/STOP (Edomain). The L526M mutation was detected only during a peak of HBV DNAimmediately after transplantation and was not detected in subsequentisolates from this patient. Nine of the 12 unique polymerase mutationsresulted in an altered HBsAg in the overlapping reading frame as shownin parentheses, L423L/F (C69C/F/S/Y), A432V (R78L), R466K (G112R), N477T(T123P), N485N/K (N131N/I/T/S), G498E (D144N), T530S (L176V), N572K(1218N) and F573Y (F2191).

There were 11 unique changes in the HBsAg post treatment when comparedto the sequences listed by Norder et al. (20) and the individual'spretreatment isolate. These were C69C/S/F/Y, R78L, T123P, T131N/I/T/S,D144N, C147S/Y, S167L, L173R, L176V, 1218N and F2191. The changes atC147S/Y, S167L and L173R did not result in a change in the overlappingpolymerase reading frame, whereas the other unique HBsAg mutations allresulted in a change in the polymerase (listed above). The T131N/I/T/Smutation has previously been detected after HBIG treatment and thechanges at T123P and D144N are at the same position as other previouslyreported HBIG associated changes. These patients did not have HBVrecurrence even in the presence of these previously noted HBIGassociated variants. No amino acid sequence in this patient group wasnoted that was common to all non-recurrence patients which was notpresent in HBV isolates from patients with recurrence nor FCVnonresponder.

(B) Patients with HBV DNA Less than 90 pg/ml (LR Group).

Pre-OLT Phase

Multiple HBV isolates from transplant patients treated with HBIG onlyand not FCV were sequenced to determine the background sequencevariation over a comparable time interval in the transplantationsetting.

Of the 10 patients with low levels of viremia, serum samples wereavailable from 9 patients pre-OLT. Sequencing of these isolatesdemonstrated that there were 12 unique changes isolated from fivepatients compared to the published HBV polymerase consensus sequence.These were at S/D455P, N469D, Y494F, Y/F497L, S/F565A/S, F/V573F/L,P583T (D Domain), N/Q584S (D domain), K585K/G (D Domain), S592N (Edomain), L593L/I/V (E domain) and N594H (E domain).

In the HBsAg of these pretreatment isolates, five amino acid variantswere detected in 4 patients when compared to the sequences published byNorder et al. (20). These variants were at P/S/A120Q P/S/A120T,S/N210A/S, S/N210R/S and F219Y/F. The first two of these HBsAg changeshave been previously associated with HBIG selected changes post OLT(23).

Post-OLT Phase

(i) HBV Recurrence

Serum samples suitable for sequencing were available from only twopatients who had HBV recurrence during HBIG treatment. The HBV sequencecharacterized from these two patients revealed that there were 6 uniquechanges compared to the published consensus and the individual'spretreatment sample. These were N469D, L492S, Y494F, T496T/N, Y497L andS548S/R(C domain). The L492S, the T496T/N and the S548S/R also changedthe HBsAg at C138R, T142T/P and A194G/A, respectively. The other changesdid not alter the HBsAg. Domain A is the only conserved domain region inwhich there were no unique changes selected in HBV isolates from LRpatients post OLT whereas several changes were selected in this domainduring FCV treatment in HR HBV isolates (see Section A above).

Within the HBsAg there were 6 unique changes in two patients whencompared to the published sequences (20) and the individual=spretreatment sample. These were V96A, P120Q, C138R, P142T/P, K160K/N andA194G/A. The K160K/N mutation resulted in a change of the HBV polymeraseat I515I/L. The other changes which affected both overlapping readingframes are listed above. Five of these changes were not detected in anypretreatment sample (Table 5). The mutation at P120Q was detectedpretreatment, and has previously been reported to be selected after HBIGtreatment (23).

(ii) HBV Non-Recurrence

Sera was available from five out of the six patients post-OLT withoutHBV recurrence. In two of these patients there were 5 changes in thepolymerase gene compared to the published consensus and the individual'spretreatment isolate. These were at L/S/R563R/C, L581L/F(D domain),L581L/Stop (D domain) and P583P/R (D domain) and L593L/I (E Domain).Only the L/S/R563R/C mutation altered the HBsAg in the overlappingreading frame at I208I/M.

The I208I/M was the only unique change detected in the HBsAg sequencescompared to those listed by Norder et al (20) and the individual=spretreatment sample. This change has not been previously noted withvaccine or HBIG escape. The amino acid variant (P/S/A120T, a known HBIGselected variant) was detected in one patient's pretreatment isolate.This was not detected in this patients post treatment isolates and thepatient did not have recurrence.

TABLE 2 Unique HBV polymerase changes in the pretreatment isolates fromFCV responders Amino acid differences compared to the publishedCorresponding HBV polymerase consensus sequence HBsAg change L423F C69SS452A no change S/D455P no change L461V no change S462A C107W Q471LS117C H/Y 472R no change H479H/Q T/M125M/T/K/R S483T no change V488EF/Y/I134S V488E/V F/Y/I134N/Y V488G F/Y/I134V, M/K/L133T R/W499G/WD/A144G I508V no change I533L no change V/L/M533I/M After stop codonV/G560E Y/F/H/C206N V/G560P K/N/S204R Q/E561S S/G/H/N/D/T207R Q/E561Q/Stop no change S/F565A S/N210R T/A568S I/L/M213F N/Q584S After HBsAGStop N/Q584H After HBsAG Stop S592H After HBsAG Stop N594H After HBsAGStop N594H/Y After HBsAG Stop M596T/M After HBsAG Stop

TABLE 3 Unique HBV HBsAg changes in the pretreatment isolates from FCVresponders Amino acid differences compared to the Correspondingpublished HBV HBsAg sequences HBV polymerase change. P67Q no change C69SL423F R73P no change R79H no change L94STOP no change V96A no changeQ101R no change C107W S462A S117C Q417L P/S/A120S/A/T/PT/P/N/I474I/T/N/S P/S/A120T no change P/S/A120T/P no changeT/M125M/T/K/R H479Q M/K/L133T V488G F/Y/I134N/Y V488V/E F/V/I134V V488GF/Y/I134S V488E F/Y/I134N V488E S/T143M no change D/A144E/N R/W499G/WA/G166V no change K/N/S204R no change Y/F/H206N V/G560E S/G/H/N/D/T207RQ/E561S S/N210R S/F565A I/M/L213F T/A568S P214L no change P217L nochange

TABLE 4 Summary of amino acid changes in HBV variants isolated from FCVnon-responders compared to the published consensus sequence (18) Aminoacid differences compared to the published Corresponding Amino acid HBVpolymerase HbsAg change in other consensus sequence change patientgroups H436N/H no change H436H/Y(3-6^(a), 27-3^(a)) S463S/Y L1091/L notdetected V537V/I C/W182Y/Stop not detected V/G560E Y206N V560E(15-1^(b))S/F565A/S S210R/S S/F 565A S/F 565 A (4-1^(b), 10-1^(b), 18-1^(b))N/Q584H After end HbsAg N/Q584H(15-1^(b)) N/Q584 S (2-3^(a), 3-1^(b),26-1^(c)) N/S/H 584N/K(3-3^(a)) K587R After end HBsAg not detected N594HAfter end HbsAg N594H (2-3^(a), 14-1^(b), 15-1^(b), 17-1^(b), 26-1^(c),31-1^(c)) N594N/Y(2-1^(b)) Amino acid changes in bold were not detectedin patients who responded to FCV ^(a)= HBV isolated from patients withHBV recurrence during FCV treatment ^(b)= HBV isolated from apretreatment isolate from a FCV treated responder ^(c)= HBV isolatedfrom a pretreatment isolate from an HBV low replicator not treated withFCV

TABLE 5 Summary of amino acid differences in HBV variants isolatedduring HBV recurrence from FCV treated patients compared to thepublished consensus Amino acid dffferences compared to the published HBVpolymnerase consensus Corresponding Amino acid change in sequence HbsAgchange other patient groups L423L/M/V I68I/M L423F/L(15-2^(a))L423F(12-1^(e)) L423L/F C69F/L L423F/L(15-2^(a)) L423F(12-1^(e)) H436H/Yno change H436N(32-2^(b)) H436Y DEL 471-474 117-120 not detected S483Tno change S483T(2-1^(e)) W499E D144E/G145R R499K(1-2^(c)) I508V nochange I508V(2-1^(e)) V519L E164D V519L(1-3^(c)) L526M no changeL526M(1-3^(c), 6-2^(a,g)) S565A S210R S565A (4-1^(e), 10-1^(e),18-1^(e)) N584S after HBsAg stop N/Q584S(3-1^(e), 26-1^(c))N584H(15-1^(f), 32-1^(b)) N/S/H584N/K after HBsAg stop N/Q584S(3-1^(e),26-1^(c)) N584H(15-1^(f), 32-1^(b)) R588R/K after HBsAg stop notdetected N594H after HBsAg stop N594H (14-1^(e), 15-1^(e), 17-1^(e),24-1^(b), 25-1^(b), 26-1^(f), 31-1^(e)) N594N/Y(2-1^(e)) Amino acidchanges in bold were not detected in patients who responded to FCV, norin patients with HR HBV who did not have HBV recurrence post-OLT. ^(a)HRFCV Responder pre-OLT, non-recurrence post OLT ^(b)HR non-responder^(c)LR with recurrence post OLT, treated with FCV post recurrence ^(d)HRnon-recurrence ^(e)HR FCV responder pre-OLT ^(f)LR pre-OLT^(g)Transitory change

TABLE 6 Unique changes in HBsAg in patients with HBV recurrencecomparison to Norder et al.(20) and all HBsAg pretreatment sequences HBVpolymerase HBsAg change Isolate FCV treatment equivalent I68I/M 5-2FCB + HBIG L423L/M/V DEL 117-120 4-3 FCB + HBIG DEL 471-474 D144E 4-3FCB + HBIG W499E G145R 4-3 FCB + HBIG W499E E164D 27-3, 4, 5 FCV + HBIGV/G519L V96A 26-3 HBIG no change C138R 26-3 HBIG L492S P142T/P 28-4 HBIGT496T/N K16OK/N 26-4 HBIG I515I/L A194G/A 26-3 HBIG S/A548S/R

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1. A method for determining the potential for a mutated HBV to exhibitreduced sensitivity to a nucleoside analogue, said method comprisingisolating DNA or corresponding mRNA from said mutated HBV and screeningfor one or more of the following mutations in the HBV DNA polymeraseN584S, N584K, S/H584N/K, R588K, N/Q 584H, K587R, wherein the notationdefines a codon number position of the HBV genotype with reference tocodon 550 being methionine in the YMDD motif, the letter(s) proceedingthe number in the notation being at least one wild-type or pre-mutationamino acid at said codon number position, and the letter(s) subsequentto the number in the notation being at least one post-mutation aminoacid at said codon number position, wherein the presence of such amutation is an indication of the likelihood of resistance to saidnucleoside analogue.
 2. A method for determining whether an HBV isolateencodes a variant DNA polymerase, said method comprising determining theamino acid sequence of its DNA polymerase directly or via a nucleotidesequence and comparing same to the amino acid sequence below:Z₄₇ Z₄₈ G I H L N P Z₄₉ K T K R(SEQ ID NO:9) wherein: Z₄₇ is S or D; Z₄₈is L or V; Z₄₉ is N or Q, wherein the variant comprises a mutation inthe HBV DNA polymerase selected from one or more of N584S, N584K,S/H584N/K, R588K, N/Q 584H, K587R, wherein the notation defines a codonnumber position of the HBV genotype with reference to codon 550 beingmethionine in the YMDD motif, the letter(s) proceeding the number in thenotation being at least one wild-type or pre-mutation amino acid at saidcodon number position, and the letter(s) subsequent to the number in thenotation being at least one post-mutation amino acid at said codonnumber position.